Species-specific PCR method for identification of Streptococcus downei

AIMS: To establish a rapid method to differentiate Streptococcus downei and S. sobrinus by multiplex PCR. METHODS AND RESULTS: A PCR primer pair specific to S. downei was designed on the basis of the nucleotide sequence of the dextranase gene of S. downei NCTC 11391T. The primer pair specifically detected S. downei, but none of the other mutans streptococci (16 strains of six species). The PCR procedure was capable of detecting 1 pg of genomic DNA purified from S. downei NCTC 11391 and as few as 14 CFU of S. downei cells. The mixture of primer pairs specific to each S. downei (this study) and S. sobrinus (Igarashi et al. 2000) detected only the strains of these two species among all the mutans streptococcal strains, and concomitantly differentiated the two species by species-specific amplicons of different lengths. CONCLUSIONS: The present PCR method is highly specific to S. downei and is useful for detection and identification of S. downei. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiplex PCR using dextranase gene primers is a useful method for simultaneous detection and differentiation of S. downei and S. sobrinus.     

Sequence Analysis of the Streptococcus mutans Ingbritt dexA Gene Encoding Extracellular Dextranase

The complete nucleotide sequence (3,747 bp) of the dextranase gene (dexA) and flanking regions of the chromosome of Streptococcus mutans Ingbritt (serotype c) were determined. The open reading frame for dexA was 2,550 bp, ending with a stop codon TGA. A putative ribosome-binding site, promoter preceding the start codon, and potential stem-loop structure were identified. The presumed dextranase protein (DexA) consisting of 850 amino acids was estimated to have a molecular size of 94,536 Da and a pI of 4.79. The nucleotide sequence and the deduced amino acid sequences of S. mutans dexA exhibited homologies of 57.8% and 47.0%, respectively, to those of Streptococcus sobrinus dex. The homologous region of dex of S. sobrinus was in the N-terminal half. The C terminus of DexA consisted of a hexapeptide LPQTGD, followed by 7 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged hexapeptide tail, which have been reported as a common structure of C termini of not only the surface-associated proteins of Gram-positive cocci but also the extracellular enzymes such as β-fructosidase of S. mutans and dextranase of S. sobrinus. The DexA protein had no significant homology with the glucosyltransferases, the glucan-binding protein, or the dextranase inhibitor of mutans streptococci.     

Characterization of the Dextranase Gene (dex) of Streptococcus mutans and Its Recombinant Product in an Escherichia coli Host

The gene (dex), which encodes the Streptococcus mutans dextranase (Dex), was cloned in Escherichia coli. The E. coli host harboring a recombinant plasmid (pSD2) containing an 8-kb BamHI insert produced a Dex protein of 133 kDa as well as smaller enzymes of 118, 104, and 88 kDa. The Dex produced by the recombinant E. coli was apparently located in the cytoplasmic fraction, not in the periplasmic nor the extracellular fractions. Subcloning and deletion analysis of pSD2 showed that the structural gene of Dex was encoded by a 4-kb BamHI-SalI fragment. The fragment also contained the dex promoter which was effective in the E. coli cell.     

Engineered dextranase from Streptococcus mutans enhances the production of longer isomaltooligosaccharides

Herein, we investigated enzymatic properties and reaction specificities of Streptococcus mutans dextranase, which hydrolyzes α-(1→6)-glucosidic linkages in dextran to produce isomaltooligosaccharides. Reaction specificities of wild-type dextranase and its mutant derivatives were examined using dextran and a series of enzymatically prepared p-nitrophenyl α-isomaltooligosaccharides. In experiments with 4-mg·mL^?1 dextran, isomaltooligosaccharides with degrees of polymerization (DP) of 3 and 4 were present at the beginning of the reaction, and glucose and isomaltose were produced by the end of the reaction. Increased concentrations of the substrate dextran (40 mg·mL^?1) yielded isomaltooligosaccharides with higher DP, and the mutations T558H, W279A/T563N, and W279F/T563N at the ?3 and ?4 subsites affected hydrolytic activities of the enzyme, likely reflecting decreases in substrate affinity at the ?4 subsite. In particular, T558H increased the proportion of isomaltooligosaccharide with DP of 5 in hydrolysates following reactions with 4-mg·mL^?1 dextran.Abbreviations CI: cycloisomaltooligosaccharide; CITase: CI glucanotransferase; CITase-Bc: CITase from Bacillus circulans T-3040; DP: degree of polymerization of glucose unit; GH: glycoside hydrolase family; GTF: glucansucrase; HPAEC-PAD: high performance anion-exchange chromatography-pulsed amperometric detection; IG: isomaltooligosaccharide; IGn: IG with DP of n (n, 2?5); PNP: p-nitrophenol; PNP-Glc: p-nitrophenyl α-glucoside; PNP-IG: p-nitrophenyl isomaltooligosaccharide; PNP-IGn: PNP-IG with DP of n (n, 2?6); SmDex: dextranase from Streptococcus mutans; SmDexTM: S. mutans ATCC25175 SmDex bearing Gln100?Ile732     

右旋糖酐发酵液酶解制备右旋糖酐40中氮的控制

右旋糖酐发酵液经过吸附除杂和陶瓷膜过滤后处理,在右旋糖酐酶的作用下降解制备右旋糖酐40,对氮进行过程控制,达到右旋糖酐40国家标准。首先以蛋白质作为检测目标,对右旋糖酐发酵液进行吸附条件研究,利用最佳吸附条件进行右旋糖酐发酵液的吸附除杂,氮的去除率达到57%以上,氮含量降至0.09%以下;然后通过陶瓷膜过滤将右旋糖酐与果糖等杂糖分离,并进一步去除氮,氮的去除率达到45%以上,氮含量降至0.040%以下;最后利用右旋糖酐酶对右旋糖酐发酵液处理液进行酶解,右旋糖酐酶解液经过乙醇分级沉淀分离制备右旋糖酐40,氮的去除率达到89%以上,氮含量降至0.003%以下,达到≤0.007%的国家标准。右旋糖酐发酵液酶解制备右旋糖酐40工艺过程氮的控制可以使氮去除率达到98%以上,产品氮含量达到国家标准(≤0.007%),分子量分布有所改善,重均分子量35 000以上,10%小分子＞7 000。     

葡聚糖酶高产菌株的诱变筛选及其摇瓶发酵条件的初步研究

从土壤中分离出的一株产葡聚糖酶酶活31 U／ml的野生菌株,经UV、^60Co、LiCl诱变筛选后得到了产葡聚糖酶高产菌株SB126,经发酵条件优化试验后检测其酶活达到85U／ml,较野生菌株提高了近两倍。葡聚糖酶摇瓶较适发酵条件为：装量30ml(250m1)、转速180r／min、pH7．0、温度30℃、接种量8％、发酵周期5d。     

一株产耐热右旋糖酐酶真菌的筛选、鉴定及其酶学性质

【目的】从土壤中筛选得到1株产耐热右旋糖酐酶的真菌。【方法】采用营养缺陷型培养基,结合稀释涂布法和平板透明圈法分离筛选出产耐热右旋糖酐酶的菌株。通过观察菌落形态、菌体形态和培养特征,结合ITS r DNA序列分析对菌株进行鉴定。研究菌株所产右旋糖酐酶的酶学性质。【结果】通过筛选得到1株产耐热右旋糖酐酶的菌株DG001,经鉴定为淡紫拟青霉(Paecilomyces lilacinus)。菌株DG001所产右旋糖酐酶的最佳催化条件为55°C,p H 5.0;最适底物为5%Dextran T70。酶在60°C以下和p H 4.0–7.0之间稳定。urea、Mn~(2+)和Mg~(2+)对酶活均有促进作用,低浓度的Mn~(2+)和urea可使酶活分别提高到116.91%和110.14%,而Cu~(2+)则对其有强烈抑制作用。该酶水解右旋糖酐T2000的产物主要是异麦芽糖和异麦芽三糖,被确定为内切右旋糖酐酶。酶对底物的亲和性随底物分子量的增加而增强。【结论】成功筛选获得1株产耐热右旋糖酐酶的菌株DG001,所产酶在较宽温度范围内具有较高活力,热稳定性好。该酶在制糖工业及不同分子量右旋糖酐的制备中具有很好的应用前景。     

交替假单胞菌LP621菌株产右旋糖苷酶的培养条件优化

从江苏连云港海域分离和筛选到1株产右旋糖苷酶的海洋细菌交替假单胞菌Pseudoalteromonas tetraodonis LP621,通过单因素试验和正交试验对该菌株产右旋糖苷酶培养条件进行优化。单因素试验结果表明,最佳培养时间为24 h,最适产酶温度为25℃;产酶pH范围为5.0~11.0,最适产酶pH为6.0;产酶NaCl浓度范围为1%~10%,NaCl浓度为4%时产酶较高;装液量在25%。麦芽糖、胰蛋白胨和酵母膏促进产酶。利用响应面方法对LP621产右旋糖苷酶的发酵条件进行优化。选择培养基pH、时间、麦芽糖浓度和装液量4因素进行优化,结果为pH 7.07,发酵时间21.94 h,麦芽糖浓度0.42%,装液量为21.88%,酶活为270.1U/mL。     

Bacillus polymyxa stimulates increased Rhizobium etli populations and nodulation when co-resident in the rhizosphere of Phaseolus vulgaris

Abstract A Gram-negative sporulating thermophilic anaerobe, designated AB11Ad, was isolated from the heated waters of the Great Artesian Basin of Australia. It grew on a variety of carbohydrates including glucose, starch, and dextran and produced a thermostable and thermoactive extracellular endo-dextranase. The enzyme was produced more actively under pH controlled continuous culture conditions than under batch conditions. Ammonium sulfate precipitated crude dextranase exhibited a temperature optimum of 70 °C and a pH optimum between 5 and 6. The half life was ~ 6.5 h at 75 °C and 2 h at 80 °C at pH 5.0 and in the absence of added dextran. 16S rRNA sequence analysis indicated that isolate AB1 lAd was a member of the genus Thermoanaerobacter .     

A comparison of α- and β-glucanase activities in fourteen species of marine molluscs

Fourteen species of marine molluscs collected from Naples Bay were compared in terms of α-amylase, dextranase, cellulase and laminarase activities. The distribution of these glucanase activities, in general, seems to be unrelated with the feeding habits of the species examined. However, the presence of both the β-glucanase activities in most of the carnivorous species strengthens the view of a common origin from a remote herbivorous stock.